Intestinal tight junction dysfunction occurs in a variety of clinical conditions, including food allergies, infections of the gastrointestinal tract, autoimmune diseases, and inflammatory bowel diseases. Healthy, mature gut mucosa with its intact tight junction serves as the main barrier to the passage of macromolecules. During the healthy state, small quantities of immunologically active proteins cross the gut host barrier. These proteins are absorbed across the mucosa through at least two pathways. The vast majority of absorbed proteins (up to 90%) cross the intestinal barrier via the transcellular pathway, followed by lysosomal degradation that converts proteins into smaller, non-immunogenic peptides. Other proteins are transported as intact proteins, through the paracellular pathway, which involves a subtle but sophisticated regulation of intercellular tight junctions that lead to protein (antigen) tolerance. When the integrity of the tight junction system is compromised, as with prematurity or after exposure to radiation, chemotherapy, and/or toxins, a deleterious response to environmental antigens (including autoimmune diseases and food allergies) can occur.
To meet the many diverse physiological and pathological challenges to which epithelia are subjected, the tight junctions or zonula occludens (ZO) must be capable of rapid, physiologic, reversible, transient, energy dependent, and coordinated responses that require the presence of a complex regulatory system.
Zonula occludens toxin, which is produced by Vibrio cholerae, has been characterized by Fasano et al., in Proc. Natl. Acad. Sci., USA, 8:5242-5246 (1991) and the sequence has been determined (GenBank accession no. A43864). The ZOT protein of Vibrio cholerae phage CXTΦ exploits the physiological mechanisms of tight junction regulation. ZOT possesses multiple domains that allow a dual function of the protein as a morphogenetic phage peptide for the Vibrio cholerae phage CTXΦ and as an enterotoxin that modulates intestinal tight junctions. When tested on rabbit ileal mucosa, zonulin occludens toxin (ZOT) increased the intestinal permeability by modulating the structure of intercellular tight junctions.
It has been found that ZOT is capable of reversibly opening tight junctions in the intestinal mucosa, and thus ZOT, when co-administered with a therapeutic agent, is able to effect intestinal delivery of the therapeutic agent, when employed in an oral dosage composition for intestinal drug delivery (WO 96/37196; and U.S. Pat. No. 5,665,389; and Fasano et al., J. Clin. Invest., 99:1158-1164 (1997). In U.S. Pat. No. 5,864,014, a ZOT receptor has been identified and purified from an intestinal cell line, i.e., CaCo2 cells. Further, in U.S. Pat. No. 5,912,323, ZOT receptors from human intestinal, heart and brain tissue have been identified and purified.
ZOT mediates a cascade of intracellular events by interacting with the surface of enteric cells. ZOT binding varies within regions of the small intestine, being detectable in the jejunum and distal ileum, decreasing along the villous-crypt axis, and not being detectable in the colon. This binding distribution coincides with the regional effect of ZOT on intestinal permeability.
Mammalian proteins that are immunologically and functionally related to ZOT have been identified. In U.S. Pat. No. 5,945,510, novel mammalian proteins that are immunologically and functionally related to ZOT and also function as the physiological modulator of mammalian tight junctions, have been identified and purified. These mammalian proteins, referred to as “zonulin”, function as the physiological effector of mammalian tight junctions.
Tight junction agonists (e.g., agonists of ZOT and/or zonulin) as contemplated herein have been identified that bind to ZOT receptor. These agonists rapidly open tight junctions in a reversible and reproducible manner, and thus can be used to facilitate the intestinal bioavailability of therapeutic or immunogenic agents in the same manner as ZOT is used as an intestinal delivery enhancer, as described in the following patent references: WO 05/010022, WO 96/37196; U.S. Pat. No. 5,827,534; U.S. Pat. No. 5,665,389; and U.S. Pat. No. 5,908,825.
Tight junction antagonists (e.g., antagonists of ZOT and/or zonulin) as contemplated herein have been identified that bind to ZOT receptor, yet do not function to physiologically modulate the opening of mammalian tight junctions. See, U.S. Pat. No. 6,458,925. The peptide antagonists competitively inhibit the binding of ZOT and zonulin to the ZOT receptor, thereby inhibiting the ability of ZOT and zonulin to physiologically modulate the opening of mammalian tight junctions. Inhibiting the opening of tight junctions in various anatomical barriers may be useful in the treatment of autoimmune diseases.